Abstract We performed a systematic identification of microRNAs (miRNAs) involved in the control of innate immunity. We identified 7 novel miRNA mutants with altered survival, colony forming in the body, and expression pattern of putative antimicrobial genes after Pseudomonas aeruginosa infection. Loss-of-function mutation of mir-45, mir-75, mir-246, mir-256, or mir-355 induced resistance to P. aeruginosa infection, whereas loss-of-function mutation of mir-63 or mir-360 induced susceptibility to P. aeruginosa infection. DAF-2 in the insulin signaling pathway acted as a target for intestinal mir-355 to regulate innate immunity. mir-355 functioned as an important link between p38 MAPK signaling pathway and insulin signaling pathway in the regulation of innate immunity. Our results provide an important molecular basis for further elucidation of the functions of various miRNAs in the regulation of innate immunity.